This invention relates to a previously commercialized Lepidopteran resistant cotton plant event, known as MON531, expressing a chimeric form of a Cry1A insect inhibitory protein. Cotton plants are susceptible to insect infestation in all areas of the world in which the plants are cultivated. Recombinant DNA technology has been applied to cells of the cotton plant for about a decade, and cotton plants which exhibit improved characteristics as a result of the insertion of heterologous DNA sequences have been produced using recombinant DNA technology since about the early 1990's. Some of the improvements exhibited by recombinant cotton plants includes herbicide tolerance, improved fiber characteristics, and resistance to insect infestation.
The first recombinant cotton plants protected from Lepidopteran insect infestation were produced, approved by regulatory agencies for commercial distribution, and subsequently commercialized in 1996. These cotton plants contained a DNA sequence encoding a chimeric Cry1A lepidopteran insect inhibitory protein, primarily from the MON531 event. This particular trait, along with an adjacent linked DNA sequence encoding a selectable marker, has been transferred by conventional breeding into a number of cotton varieties, each of which are particularly suited for enhanced production of cotton in diversified geographic locations throughout the world. These recombinant varieties have enjoyed a tremendous commercial success for a number of reasons. One reason is that yields per acre of cotton production have on average improved dramatically because of reduced insect infestation as a result of the presence of the insect inhibitory protein present within each cell of the cotton plant. Another principle reason for the commercial success has been the reduced labor and expense due to the reduction in applications of chemical pesticides required to protect the crop from insect infestation. In addition, the reduction in chemical pesticide applications improves the overall health of the environment by avoiding the annihilation of insects or arachnids and other species which present no material threat to the crop in the field, reduces the load of chemical pesticide toxins applied to the environment, and allows the farmer to avoid the risks associated with the potentially harmful effects of exposure to chemical pesticides.
It soon became apparent that a product with a broader range of efficacy against insects would be desirable. While it may be seen as simple in view of the chemical arts to provide a combination of insect inhibitory proteins for this purpose and possibly to delay or prevent toxin resistance from being developed in the target insect population, in reality the development of a plant meeting these characteristics is problematic, and requires a great deal of resources, technical ability, and trial and error experimentation in order to obtain a single recombinant plant transformation event which results in a morphologically normal plant exhibiting the desired combination of insect inhibitory proteins produced in sufficient levels and at appropriate times during the plant growing season and in the tissues upon which target pest species feed.
Thus, there existed a need for the development and characterization of cotton plants exhibiting the characteristics of enhanced insect resistance as a result of the presence of two or more insect inhibitory proteins produced from DNA sequences incorporated into the genome of the plant cells. Furthermore, it would be desirable for the insect inhibitory traits (i) to segregate independently of one another, (ii) to not cause any adverse effects upon the physiology and metabolism of the plant, and (ii) to have little if any adverse effect upon the yield or quality of the fiber produced from said plant.
It is advantageous to be able to detect the presence of a particular event in order to determine whether progeny of a sexual cross contain a transgene of interest. In addition, a method for detecting a particular event may be helpful for complying with regulations requiring the pre-market approval of the sale of seeds to produce transgenic crop plants and foods derived from such plants, for example, or for use in environmental monitoring, monitoring traits in crops in the field, or monitoring products derived from a crop harvest, as well as for use in ensuring compliance of parties subject to regulatory or contractual terms.
It is possible to detect the presence of a transgene by any nucleic acid detection method known in the art including but not limited to thermal amplification (PCR™) or DNA hybridization using nucleic acid probes. Typically, for the sake of simplicity and uniformity of reagents and methodologies for use in detecting a particular DNA construct that has been used for transforming various plant varieties, these detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc., because for many DNA constructs, the coding sequence region is interchangeable. As a result, such methods may not be useful for discriminating between separate events produced from the same DNA construct or very similar constructs. These methods can be used, however, if the sequence of chromosomal DNA adjacent to the inserted DNA (“flanking DNA”) is known. An event-specific thermal amplification (PCR™) assay is discussed, for example, by Windels et al. (Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identified glyphosate tolerant soybean event 40-3-2 using a thermal amplification primer set spanning the junction between the insert and flanking DNA. Specifically, one primer included sequence from within the insert and a second primer included sequence from flanking DNA. Such a method was also developed for event MON531 and is the subject of a separate patent application. It would be desirable to have such a method that would detect the presence of the new event of the present invention, even in the presence of event MON531. These and other advantageous advances have been achieved by the present invention.